RSV causes serious lower respiratory tract disease in infants and young children. RSV has a non-segmented, negative-sense RNA genome and codes for two non-structural proteins, the NS1 and NS2. Previous studies indicated that the NS2 may have a role in CTL response. More recent studies indicate that NS1 probably acts as a negative regulator for RSV replication. However, the biochemical basis for these effects is not known. In this project, I would like to define the structural domains that bring about these functional effects. With this goal in mind the following studies are being carried out. Overlapping peptides along the entire sequence of the NS1 and NS2 were made by chemical synthesis. Polyclonal antibodies to the N-terminal and C-terminal peptides of the NS1 and NS2 were also raised in rabbits to define the naturally occuring forms of these proteins. Future studies include the development of in vitro transcription and replication aasys of RSV-infected cell lysates in which various peptides either singly or in combination will be tested for their inhibitory potential. These results will identify the domains in the natural protein sequences that are responsible for their negative regulatory function. The specificty of these effects will be further tested in the presence of specific antibodies. In addition, these reagents will identify the other viral gene products involved in the inhibition. The inhibitory peptides will be studied for their structure-function relationships to design better negative regulators. In addition, cell lines will be transformed permanently with these NS1 and NS2 genes and the expression of these proteins will be determined. Once expressor cell lines identified, they will be infected with RSV and the effect of the expressed protein on RSV growth and replication will be investigated. Moreover, using yeast two-hybrid system, interaction of these NS1 and NS2 proteins with host proteins will be investigated.